Microorganism producing 5&#39;-xanthylic acid

ABSTRACT

The invention relates to  Corynebacterium ammoniagenes  CJXSP 0201 KCCM 10448 producing 5′-xanthylic acid. More specifically, the invention relates to  Corynebacterium ammoniagenes  CJXSP 0201 KCCM 10448 which is a mutant strain of  Corynebacterium ammoniagenes  KCCM 10340 having a resistance to osmotic pressure. In order to obtain mutant strain having enhanced respiratory activity, the present invention adopted  Corynebacterium ammoniagenes  KCCM 10340 as parent strain and treated it with UV radiation and mutation derivatives such as N-methyl-N′-nitro-n-nitrosoguanidine (NTG) according to ordinary procedure. Therefore,  Corynebacterium ammoniagenes  CJXSP 0201 KCCM 10448 of the present invention makes it possible to overcome growth-standing phase rapidly on early culture and for same period of fermentation, can accumulate 5′-xanthylic acid in culture medium at a high yield and concentration rate.

TECHNICAL FIELD

The invention relates to a microorganism producing 5′-xanthylic acid.More particularly, the invention relates to selecting a mutant strain ofCorynebacterium ammoniagenes KCCM 10340 in order to enhance growthactivity, which can rapidly overcome growth-standing phase on earlyculture and can accumulate 5′-xanthylic acid in culture medium for sameperiod of fermentation at a high yield and high concentration rate.

BACKGROUND ART

5′-xanthylic acid is an intermediate in the nucleic acid biosynthesisprocess, which is physiologically important in the body of animals andplants, used in food, medical supplies and other various field. Theinvention relates to a mutant strain from a known strain Corynebacteriumammoniagenes KCCM 10340, producing 5′-xanthylic acid by a directfermentation method at a high yield and high concentration rate thanexisting technique.

5′-xanthylic acid is an intermediary product of purine nucleotidebiosynthesis process and important material for producing 5′-guanylicacid. A widely used method to produce 5′-guanylic acid having finenessand high quality is microorganism fermentation method which produces5′-xanthylic acid first and converts it into 5′-guanylic acidenzymatically, therefore, to produce 5′-guanylic acid, correspondingamount of 5′-xanthylic acid is necessary. Conventional methods toproduce 5′-xanthylic acid are chemosynthesis, deaminization of5′-guanylic acid which is produced as a result of decomposition ofribonucleic acid in yeast, a fermentation method to add xanthine asprecursor material in fermenting medium, a fermentation method to use amutant strain of microorganism, a method to add antibiotic material (JP1477/42 and JP 20390/44), a method to add surfactant (JP 3825/42 and JP3838/42) and so on. Among these, a direct fermentation method of5′-xanthylic acid by a mutant strain of microorganism is quiteadvantageous in terms of industrial aspect. Thus, we inventors developeda mutant strain with increased productivity of 5′-xanthylic acid, bymodifying the existing character of Corynebacterium ammoniagenes KCCM10340 into the character of producing 5′-xanthylic acid at a large yieldrate.

Most microorganisms reach to the condition that the volume doesn'tincrease any more when keep on culturing under the constant condition,and especially the concentration of microorganism producing primarymetabolite, a growth-dependent product, doesn't increase any more. It ismainly caused by limited supply of dissolved oxygen. Method of enhancingaeration and agitation condition, for removal of the limited supply ofdissolved oxygen, is used, but there is technical and economical limitin actual production method. To overcome the limit and increase yieldrate and concentration of 5′-xanthylic acid by enhancing the volume ofmicroorganism and various physiological activity, under the limitedsupply of dissolved oxygen, we inventors thought that the method ofproducing the novel microorganism showing superior growth under the samecondition, would be useful. Thus, the inventors investigated and foundout that a mutant strain, which can rapidly overcome growth-standingphase on early and shows superior growth, is most effective, and canproduce 5′-xanthylic acid at a high yield and high concentration ratethan existing technique, and accomplished in this invention.

DISCLOSURE OF THE INVENTION

The invention relates to Corynebacterium ammoniagenes CJXSP 0201(KCCM-10448) which is a mutant strain of Corynebacterium ammoniagenesKCCM 10340, producing 5′-xanthylic acid.

The CJXSP 0201 is obtained by adapting Corynebacterium ammoniagenes KCCM10340 as parent strain by spontaneous mutation method and selecting amutant strain from them. The spontaneous mutation method is thefollowing. 5 mL of nutrient medium (glucose 20 g/L, peptone 10 g/L,yeast extract 10 g/L, sodium chloride 2.5 g/L, urea 3 g/L, adenine 150mg/L, guanine 150 mg/L, pH 7.2) was poured into a test tube havingdiameter of 18 mm and sterilized under pressure according to the commonmethods. Then, Corynebacterium ammoniagenes KCCM 10340 was seeded intoand it was cultured with shaking at 200 rpm, 30° C. for 18 hours and theresultant was used as seed culture. 50 μl of the seed culture was seededinto 500 mL-Erlenmeyer flask for shaking which had been sterilized and40 mL of minimum medium (glucose 20 g/L, potassium phosphate monobasic 1g/L, potassium phosphate dibasic 1 g/L, urea 2 g/L, ammonium sulfate 3g/L, magnesium sulfate 1 g/L, calcium chloride 100 mg/L, ferrous sulfate20 mg/L, manganese sulfate 10 mg/L, zinc sulfate 10 mg/L, biotin 30μg/L, thiamine hydrochloride 0.1 mg/L, copper sulfate 0.8 mg/L, adenine20 mg/L, guanine 20 mg/L, pH 7.2) had been added in. Then, it wascultured with shaking at 200 rpm, 30° C. for 24 hours, and when itreached to early log phase of growth, 50 μl of the culture was seededinto another 500 mL-Erlenmeyer flask for shaking in which 40 mL of theminimum medium had been added. And when it reached to early log phase ofgrowth (Optical Density 0.5 (λ=562 nm)) again, 50 μl of the culture wasseeded into another 500 mL-Erlenmeyer flask for shaking in which theminimum medium had been added again. Such a process, namely subculturewas repeated 20 times. The final culture was streaked on petri-dish ofthe minimum medium containing 1.5% agar and was cultured in 30° C.incubator until colony formed. Among the colonies, colonies showingrapid growth rate relatively were selected as superior mutant strain.And from them, a strain which shows superior 5′-xanthylic acidproductivity and growth rate, was separated, named CJXSP 0201, and itwas deposited under Budapest Treaty to the Korean Culture Center ofMicroorganisms on Nov. 21, 2002 with accession Number KCCM 10448. Thetime for colony forming is 38 hours in KCCM 10340, a known strain, whileCJXSP of the invention 0201 takes 31 hours, therefore CJXSP is a mutantstrain having character of superior growth.

BEST MODE FOR CARRYING OUT THE INVENTION EXAMPLE 1

Used strains: Corynebacterium ammoniagenes KCCM 10340, Corynebacteriumammoniagenes CJXSP 0201

Seed medium: glucose 30 g/L, peptone 15 g/L, yeast extract 15 g/L,sodium chloride 2.5 g/L, urea 3 g/L, adenine 150 mg/L, guanine 150 mg/L,pH 7.2

Fermentation medium: (1) A medium: glucose 60 g/L, magnesium sulfate 10g/L, ferrous sulfate 20 mg/L, zinc sulfate 10 mg/L, manganese sulfate 10mg/L, adenine 30 mg/L, guanine 30 mg/L, biotin 100 μg/L, copper sulfate1 mg/L, thiamine hydrochloride 5 mg/L, calcium chloride 10 mg/L, pH 7.2

(2) B medium: potassium phosphate monobasic 10 g/L, potassium phosphatedibasic 10 g/L, urea 7 g/L, ammonium sulfate 5 g/L

Fermentation method: 5 mL of the seed medium was poured into a test tubehaving diameter of 18 mm and sterilized under pressure according to thecommon methods. After the sterilization, a Corynebacterium ammoniagenesKCCM 10340 and Corynebacterium ammoniagenes CJXOL 0201 were seeded inrespectively and it was cultured with shaking at 180 rpm, 30° C. for 18hours. The resultant was used as seed culture. Then, as fermentationmedium, A medium and B medium were sterilized separately under pressureaccording to the common methods and 29 mL of A medium and 10 mL of Bmedium were respectively poured into sterilized 500 mL-Erlenmeyer flaskfor shaking and 1 mL of the above-mentioned seed culture was seeded intoand fermented at 200 rpm, 30° C. for 90 hours. After the fermentationwas completed, the amount of accumulation of 5′-xanthylic acid in themedium showed that the amount in KCCM 10340 was 23.0 g/L and the amountin CJXSP 0201 was 24.7 g/L. (The concentration of accumulated5′-xanthylic acid is given by 5′-sodium xanthate.7H₂O.)

EXAMPLE 2

Used strains: same as example 1.

Primary seed medium: same as the seed medium of example 1.

Secondary seed medium: glucose 60 g/L, potassium phosphate monobasic 2g/L, potassium phosphate dibasic 2 g/L, magnesium sulfate 1 g/L, ferroussulfate 22 mg/L, zinc sulfate 15 mg/L, manganese sulfate 10 mg/L, coppersulfate 1 mg/L, calcium chloride 100 mg/L, biotin 150 μg/L, adenine 150mg/L, guanine 150 mg/L, thiamine hydrochloride 5 mg/L, antifoaming agent0.6 mL/L, pH 7.2

Fermentation medium: glucose 151 g/L, phosphoric acid 32 g/L, potassiumhydroxide 25 g/L, adenine 198 mg/L, guanine 119 mg/L, ferrous sulfate 60mg/L, zinc sulfate 42 mg/L, manganese sulfate 15 mg/L, copper sulfate2.4 mg/L, alaniate 22 mg/L, NCA 7.5 mg/L, biotin 0.4 mg/L, magnesiumsulfate 15 g/L, cystinate 30 mg/L, histidinate 30 mg/L, calcium chloride149 mg/L, thiamine hydrochloride 15 mg/L, antifoaming agent 0.7 mL/L,CSL 27 mL/L, tuna extract 6 g/L, pH 7.3

Primary seed culture: 50 mL of the primary seed medium was poured into500 mL-Erlenmeyer flask for shaking and sterilized under pressure at121° C. for 20 minutes. After cooling, Corynebacterium ammoniagenes KCCM10340 and Corynebacterium ammoniagenes CJXOL 0201 were seeded intorespectively and it was cultured with shaking at 180 rpm, 30° C. for 24hours.

Secondary seed culture: The secondary seed medium was poured into 5L-experimental fermentation baths (2 L each) and sterilized underpressure at 121° C. for 10 minutes. After cooling, 50 mL of the aboveprimary seed culture was seeded and cultured with the air supply of 0.5vvm, at 900 rpm, 31° C., for 24 hours. During the culturing process, thepH level of the medium was kept at 7.3 with adjusting by ammoniasolution.

Fermentation method: The fermentation medium was poured into 30L-experimental fermentation baths (8 L each) and sterilized underpressure at 121° C. for 20 minutes. After cooling, the above secondaryseed culture was seeded into (1.5 L each) and cultured with the airsupply of 1 vvm, at 400 rpm, 33° C. Whenever the residual sugar leveldrops below 1% during the culturing process, sterilized glucose wassupplied and the total sugar level in the fermentation medium was keptat 30%. During the culturing process, the pH level of the medium waskept at 7.3 with adjusting by ammonia solution and the process took 90hours. After the fermentation was completed, the amount of accumulationof 5′-xanthylic acid in the medium showed that the amount in KCCM 10340was 137.2 g/L and the amount in CJXSP 0201 was 146.8 g/L. (Theconcentration of accumulated 5′-xanthylic acid is given by 5′-sodiumxanthate.7H₂O.)

INDUSTRIAL APPLICABILITY

The invention adopted Corynebacterium ammoniagenes KCCM 10340 as parentstrain and treated it UV radiation, mutation derivatives such asN-methy-N′-nitro-n-nitrosoguanidine (NTG) according to ordinaryprocedure. The KCCM 10340 strain has a resistance to osmotic pressure,caused by high concentration of 5′-xanthylic acid accumulated duringculturing process, high concentration of glucose and various carbonsource added into culture medium, which results in the high osmoticpressure outside bacterial body, inhibition of normal physiologicalactivity of 5′-xanthylic acid-producing cell and decrease of5′-xanthylic acid production. In order to obtain a strain havingenhanced character of growth activity under the same condition, theinvention modified Corynebacterium ammoniagenes KCCM 10340 and selecteda mutant strain which can rapidly overcome growth-standing phase onearly culture, and made it possible to accumulate 5′-xanthylic acid inculture medium at a high yield and high concentration rate for sameperiod of fermentation.

1. Corynebacterium ammoniagenes CJXSP 0201 (Accession Number: KCCM10448) which can rapidly overcome growth-standing phase on early cultureand produce 5′-xanthylic acid.
 2. A method of producing 5′-xanthylicacid characterized by using Corynebacterium ammoniagenes CJXSP 0201(Accession Number: KCCM 10448) of claim 1.